Molecular analysis of the heavy chain variable region genes of human hybridoma clones specific for coagulation factor VIII

Summary

Hemophilia A is a X-linked hematologic disorder characterized by undetectable or low amounts of functional coagulation factor VIII (FVIII). Replacement therapy induces FVIII neutralizing antibody (Ab) (inhibitor) in a proportion of patients which makes further treatment of these patients ineffective and costly. To envisage mechanisms underlying inhibitor development, seven hybridoma clones specific for FVIII were generated from two hemophiliaA patients with high titer of inhibitor. Specificity and isotype of the monoclonal antibodies (mAbs) were determined by ELISA. Immunoglobulin (Ig) variable region heavy (V_H) chain gene family usage was identified by RT-PCR usingV_H1–6 specific primers. Nucleotide sequences of the V_H gene of FVIII specific clones were determined and aligned to the most homologous germ line genes in the GenBank. Analysis of the expressed V_H genes by RT-PCR revealed that the hybridomas utilized either the V_H1 (71%) or the V_H3 (29%) gene family. Three V_H domains were encoded by V1–69 (DP-10),V1–2 (DP-8),andV1–8 (DP-15) genes and two by V1–18 (DP-14) gene, all from the V_H1 gene family. Of the V_H3-gene family expressing clones, one belonged to V3–66 (DP-86) and the other one toV3–21 (DP-77) germline genes. The CDR3 length was found to be highly different amongst these clones ranging from 11 to 22 amino acid residues. These data suggest that FVIII-specific Abs preferentially use V_H gene segments derived from V_H1 gene family. Diversity of the expressed V_H genes and their CDR3 length implies that different epitopes are recognized by these mAbs.

Keywords

Hemophilia A - factor VIII - human hybridoma - V_H gene

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